The preparation of monolithic separation media for HPLC composed of a single, continuous polymer macroporous support has been reported since the late 1960s. Due to its unique material properties, polymer-based monolithic columns have become an attractive alternative for packed columns, especially for the LC separations of complex biomolecular samples such as proteins, oligonucleotides, and peptides.
This article describes the LC performance of 1-mm-i.d. poly(styrene-co-divinylbenzene) monolithic columns, which were evaluated for reversed-phase, gradient elution separations of intact proteins. The effects of column temperature, flow rate, and gradient time on peak width and peak capacity are discussed. High column efficiency at optimized LC conditions is demonstrated with the reversed-phase separation of a protein sample containing ribonuclease A, myoglobin, and carbonic anhydrase and the separation of a complex mixture of intact E. coli proteins.
Instrumentation and column technology
An UltiMate® 3000 Proteomics MDLC system (Dionex, Germering, Germany) equipped with a membrane degasser, x2 dual-gradient pump, thermostated flow manager, well-plate sampler, and UV detector was controlled by Chromeleon® chromatography data system software and was used for all HPLC experiments (all from Dionex, Germering, Germany). The ProSwift® RP-10R (1 × 50 mm) monolithic column (Dionex, Amsterdam, The Netherlands) was used for the reversed-phase protein separations.
Results and discussion
Optimizing the monolithic bed structure
Figure 1 - Scanning electron micrograph of the polymer morphology of the 1-mm-i.d. ProSwift RP-10R monolithic column.
Polymer-based monolithic stationary phases typically offer high-efficiency protein separations due to the absence of mesopores in the polymer backbone. Consequently, mass transfer is driven by convection instead of diffusion. In addition, the macropore and globule size of the ProSwift RP-10R monolithic column was tuned to maximize separation efficiency while operating the column at volumetric flow rates (typically around 60 µL/min) to minimize sample dilution, thus maximizing detection sensitivity. Figure 1 shows the typical polymer globular structure of the ProSwift RP-10R monolithic column. From the figure it is clear that the monolithic material is well attached (covalently) to the surface of the capillary wall. This increases the robustness of the column during prolonged use and does not require the frits used in packed columns.
High-efficiency protein separations
Peak capacity in gradient LC is defined as the maximum number of peaks that can be separated with a resolution of 1 and elute in the applied gradient window. The effects of LC conditions (e.g., column temperature, flow rate, and gradient time) on peak capacity were investigated for separations of intact proteins. At elevated column temperatures, diffusion is enhanced, leading to faster mass transfer and narrower peaks. Since the poly(styrene-co-divinylbenzene) monolithic material exhibits excellent temperature stability, the column was operated at 80 °C. With increasing gradient time, an increase in peak capacity was observed. At optimized LC conditions (flow rate in the optimum of the van Deemter curve and a column temperature of 80 °C), a steep initial increase in peak capacity from 34 to 300 was obtained when increasing the gradient time from 5 to 20 min. At longer gradient times and shallower gradients, the peak capacity leveled off to a maximum of 475 at a gradient time of 120 min. This effect is caused by a linear increase in peak width of intact proteins with longer gradient time. Although longer gradient times may yield higher peak capacities, the price to pay in analysis time becomes unfavorable and two-dimensional chromatography may be used as an alternative.
Figure 2 - High-efficiency separation of E. coli proteins applying a 60-min gradient. Other LC conditions: flow rate = 80 µL/min, column temperature = 80 °C; aqueous acetonitrile gradient (containing 0.05% TFA [trifluoroacetic acid]) from 18 to 44%.
An example of a high-efficiency separation of a complex protein extract (E. coli) is shown in Figure 2. Whereas commonly used columns for protein separations are able to differentiate between 50 proteins, the ProSwift RP-10R monolithic column easily generates a peak capacity of around 400 in an analysis time of 60 min. In addition, running the column at volumetric flow rates from 50 to 100 µL/min ensures very good compatibility with electrospray interfacing prior to mass spectrometric detection.
Ultrafast gradient separations
Figure 3 - Fast LC separation of ribonuclease A, myoglobin, and carbonic anhydrase applying a 10-min (a) and 1-min (b) gradient. Other LC conditions: a) flow rate = 60 µL/min, column temperature = 80 °C; aqueous acetonitrile gradient (containing 0.05% TFA) from 12 to 64%; b) flow rate = 100 µL/min, column temperature = 80 °C; aqueous acetonitrile gradient (containing 0.05% TFA) from 16 to 80%.
For the separation of moderately complex protein samples, the ProSwift RP column is typically operated at a flow rate of 60 µL/min and a temperature of 80 °C, and applying gradient times of 10 min. Figure 3a shows the separation of a simple protein test mixture containing ribonuclease A, myoglobin, and carbonic anhydrase. To speed up the separation, the column was operated at a high flow rate of 100 µL/min and applying 80 °C column temperature and a short gradient (Figure 3b). The separation was obtained within 1 min, yielding peak widths at half of the peak height of only 1 sec.
Conclusion
The ProSwift RP-10R column provides high-resolution, high-speed separations of intact proteins. After optimizing column temperature, flow rate, and gradient time, a peak capacity of 475 could be achieved within 2 hr. This makes the column a valuable tool for separations of complex protein mixtures as often encountered in proteomics research.
The column can also be applied for high-speed separations of relatively simple protein mixtures, as encountered in quality control laboratories, for example. One-minute protein separations were demonstrated with peak widths at half height of only 1 sec.
The authors are with Dionex Corp., Abberdaan 114, 1046 AA, Amsterdam, The Netherlands; tel.: +31 20 683 9768; fax: +31 20 685 3452; e-mail: [email protected].